The c-myc protein is a member of the helix-loop-helix/leucine zipper (HLH/LZ).sup.1 family of transcription factors that forms heterodimers with Max (1-3). In general, trans-activating Myc:Max heterodimers are found in proliferating cells, while trans-repressing Mad:Max heterodimers are found in differentiated cells. The c-myc protein level influences cell proliferation, differentiation, and neoplastic transformation, presumably by affecting the balance between Myc:Max and Mad:Max heterodimers (4). When c-myc protein is overexpressed or is induced at inappropriate times, this balance is perturbed, and cell proliferation and differentiation are disrupted. For example, c-myc overexpression prevents or delays cell differentiation (5, 6). It also blocks serum-starved cells from entering the G.sub.o phase of the cell cycle and instead induces them to undergo apoptosis (7). c-myc overexpression is also implicated in tumor formation in experimental animals and in human patients with Burkitt's lymphoma (8, 9). These and other deleterious consequences of aberrant c-myc expression highlight the importance of understanding all aspects of c-myc gene regulation.
 FNT .sup.1 The abbreviations used herein are: HLH/LZ, helix-loop-helix/leucine zipper; AURE, AU-rich element; UTR, untranslated region; CRD, coding region determinant; CRD-BP, coding region determinant-binding protein; DTT, dithiothreitol; EGTA, ethylene glycol bis(2 aminoethyl ether)-N,N' (tetraacetic acid); PMSF, phenylmethyl-sulfonylflouride; S130, post-polysomal supernatant; SDS, sodium dodecyl sulfate; RSW, ribosomal salt wash; PCR, polymerase chain reaction; bp, base pairs; EST, Expressed Sequence Tags; RACE, rapid amplification of cDNA ends; BAC, Bacterial Artificial chromosome; GCG, Genetics Computer Group; IP, immunoprecipitation; mRNP, messenger ribonucleoprotein; hnRNPK, heterogeneous nuclear ribonucleoprotein K; HRP, horseradish peroxidase; HSP-90, heat shock protein-90; MOPS, morpholinepropanesulfonic acid; KH, K homology; ORF, open reading frame; FMR, familial mental retardation; FMRP, FMR RNA-binding protein; hKOC, human KH domain protein overexpressed in human cancer; PAG, polyacrylamide gel; PAGE, polyacrylamide gel electrophoresis; ECL, enhanced chemiluminescent.
The c-myc protein is regulated by phosphorylation, protein:protein interactions, and changes in its half-life (10-12). c-myc mRNA levels are regulated transcriptionally and post-transcriptionally, and changes in c-myc mRNA stability can result in large fluctuations in c-myc protein levels. The c-myc mRNA half-life is normally only 10 to 20 minutes but can be prolonged 3- to 6-fold when necessary. For example, c-myc mRNA is relatively stable in replicating fetal rodent hepatocytes, which produce abundant c-myc mRNA. It is far less stable in non-growing adult hepatocytes, which contain little or no c-myc mRNA (13, 14). However, it is up-regulated and stabilized several-fold when adult hepatocytes replicate following partial hepatectomy (15, 16).
Two cis-acting sequence elements in c-myc mRNA contribute to its intrinsic instability and perhaps also to its post-transcriptional regulation: an AU-rich element (AURE) in the 3'-untranslated region (3'-UTR) and a 180 nucleotide coding region determinant (CRD). The CRD encodes part of the HLH/LZ domain and is located at the 3' terminus of the mRNA coding region. Four observations indicate how the c-myc CRD functions independently of the AURE to affect c-myc mRNA expression. (i) c-myc mRNA lacking its CRD is more stable than wild-type c-myc mRNA (17-20). (ii) The CRD is required for the post-transcriptional down-regulation of c-myc mRNA that occurs when cultured myoblasts fuse to form myotubes (20, 21). (iii) Inserting the c-myc CRD in frame within the coding region of .beta.-globin mRNA destabilizes the normally very stable .beta.-globin mRNA (22). (iv) The c-myc CRD is necessary for up- and down-regulating c-myc mRNA levels in transgenic mice undergoing liver regeneration following partial hepatectomy (13, 15, 16, 23-25). In summary, the c-myc CRD influences c-myc mRNA stability in animals and in cultured cells.
We have investigated c-myc mRNA stability and the function of the CRD using a cell-free mRNA decay system that includes polysomes from cultured cells. The polysomes contain both the substrates (mRNAs) for decay and at least some of the enzymes and co-factors that affect mRNA stability. Polysomes are incubated for different times in an appropriate buffer system, and the decay rates of polysomal mRNAs such as c-myc are monitored by hybridization assays. This system reflects many aspects of mRNA decay in intact cells (26-29). For example, mRNAs that are unstable in cells are also relatively unstable in vitro; mRNAs that are stable in cells are stable in vitro (26). In standard reactions, the polysome-associated c-myc mRNA was degraded rapidly in a 3' to 5' direction, perhaps by an exonuclease (29). An alternative decay pathway became activated when the reactions were supplemented with a 180 nucleotide sense strand competitor RNA corresponding to the c-myc CRD. This CRD RNA induced endonucleolytic cleavage within the c-miyc CRD, resulting in an 8-fold destabilization of c-myc mRNA (30). These effects seemed to be specific for c-myc. Other competitor RNAs did not destabilize c-myc mRNA, and c-myc CRD competitor RNA did not destabilize other mRNAs tested.
Based on these observations, we hypothesized that a protein was bound to the c-myc CRD. We further suggested that this protein shielded the CRD from endonuclease attack, that the CRD competitor RNA titrated the protein off of the mRNA, and that the unprotected c-myc CRD was then attacked by an endonuclease. Consistent with this model, we detected a protein that binds strongly in vitro to a c-myc CRD .sup.32 P-RNA probe (30). This protein, the c-myc coding region determinant-binding protein (CRD-BP), was subsequently purified to homogeneity (31). We then found that the CRD-BP is developmentally regulated, being expressed in fetal and neonatal rats but not in adult animals (32).